Abstract

Purpose: We considered the possibility that cGMP serves as a second messenger regulating the activity of ion channels in Müller cells. Methods: Whole-cell currents of freshly dissociated or cultured Müller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. RNA from cultured human Müller cells was analyzed by RT-PCR using primers for a sequence of the rod cyclic nucleotide-gated (rCNG) channel. Intracellular calcium measurements were made by microspectrofluorometry using fura-2 AM. Results: A nonselective cation current was activated in Müller cells exposed to 8-bromo cGMP or internally perfused with cGMP. This current was reversibly blocked by cadmium and minimally activated by cAMP. Our molecular analysis demonstrated that human Müller cells contain transcripts closely related to the rCNG channel. Consistent with a calcium permeability of CNG channels, exposure of fresh Müller cells to 8-bromo cGMP induced a rise in the intracellular calcium concentration by a mechanism dependent on extracellular calcium. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect on Müller cell currents of the NO donors, s-nitroprusside and s-nitrosocysteine. These NO generators activated a nonspecific current similar to that elicited by cGMP. Conclusions: Electrophysiological and molecular analyses indicate that Müller cells express cGMP-gated cation channels. NO may be one of the intercellular signals initiating the activation of these nonspecific cation channels. The CNG channels of Müller cells may significantly affect the physiology of these glia by providing pathways for an influx of calcium and for the redistribution of excess extracellular potassium.

abstract = "Purpose: We considered the possibility that cGMP serves as a second messenger regulating the activity of ion channels in M{\"u}ller cells. Methods: Whole-cell currents of freshly dissociated or cultured M{\"u}ller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. RNA from cultured human M{\"u}ller cells was analyzed by RT-PCR using primers for a sequence of the rod cyclic nucleotide-gated (rCNG) channel. Intracellular calcium measurements were made by microspectrofluorometry using fura-2 AM. Results: A nonselective cation current was activated in M{\"u}ller cells exposed to 8-bromo cGMP or internally perfused with cGMP. This current was reversibly blocked by cadmium and minimally activated by cAMP. Our molecular analysis demonstrated that human M{\"u}ller cells contain transcripts closely related to the rCNG channel. Consistent with a calcium permeability of CNG channels, exposure of fresh M{\"u}ller cells to 8-bromo cGMP induced a rise in the intracellular calcium concentration by a mechanism dependent on extracellular calcium. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect on M{\"u}ller cell currents of the NO donors, s-nitroprusside and s-nitrosocysteine. These NO generators activated a nonspecific current similar to that elicited by cGMP. Conclusions: Electrophysiological and molecular analyses indicate that M{\"u}ller cells express cGMP-gated cation channels. NO may be one of the intercellular signals initiating the activation of these nonspecific cation channels. The CNG channels of M{\"u}ller cells may significantly affect the physiology of these glia by providing pathways for an influx of calcium and for the redistribution of excess extracellular potassium.",

N2 - Purpose: We considered the possibility that cGMP serves as a second messenger regulating the activity of ion channels in Müller cells. Methods: Whole-cell currents of freshly dissociated or cultured Müller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. RNA from cultured human Müller cells was analyzed by RT-PCR using primers for a sequence of the rod cyclic nucleotide-gated (rCNG) channel. Intracellular calcium measurements were made by microspectrofluorometry using fura-2 AM. Results: A nonselective cation current was activated in Müller cells exposed to 8-bromo cGMP or internally perfused with cGMP. This current was reversibly blocked by cadmium and minimally activated by cAMP. Our molecular analysis demonstrated that human Müller cells contain transcripts closely related to the rCNG channel. Consistent with a calcium permeability of CNG channels, exposure of fresh Müller cells to 8-bromo cGMP induced a rise in the intracellular calcium concentration by a mechanism dependent on extracellular calcium. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect on Müller cell currents of the NO donors, s-nitroprusside and s-nitrosocysteine. These NO generators activated a nonspecific current similar to that elicited by cGMP. Conclusions: Electrophysiological and molecular analyses indicate that Müller cells express cGMP-gated cation channels. NO may be one of the intercellular signals initiating the activation of these nonspecific cation channels. The CNG channels of Müller cells may significantly affect the physiology of these glia by providing pathways for an influx of calcium and for the redistribution of excess extracellular potassium.

AB - Purpose: We considered the possibility that cGMP serves as a second messenger regulating the activity of ion channels in Müller cells. Methods: Whole-cell currents of freshly dissociated or cultured Müller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. RNA from cultured human Müller cells was analyzed by RT-PCR using primers for a sequence of the rod cyclic nucleotide-gated (rCNG) channel. Intracellular calcium measurements were made by microspectrofluorometry using fura-2 AM. Results: A nonselective cation current was activated in Müller cells exposed to 8-bromo cGMP or internally perfused with cGMP. This current was reversibly blocked by cadmium and minimally activated by cAMP. Our molecular analysis demonstrated that human Müller cells contain transcripts closely related to the rCNG channel. Consistent with a calcium permeability of CNG channels, exposure of fresh Müller cells to 8-bromo cGMP induced a rise in the intracellular calcium concentration by a mechanism dependent on extracellular calcium. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect on Müller cell currents of the NO donors, s-nitroprusside and s-nitrosocysteine. These NO generators activated a nonspecific current similar to that elicited by cGMP. Conclusions: Electrophysiological and molecular analyses indicate that Müller cells express cGMP-gated cation channels. NO may be one of the intercellular signals initiating the activation of these nonspecific cation channels. The CNG channels of Müller cells may significantly affect the physiology of these glia by providing pathways for an influx of calcium and for the redistribution of excess extracellular potassium.